EAGER: Collaborative Research: Tracking of KOR1 Protein Transport in Arabidopsis using Fluorescent-Timer Imaging System

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In this project, methods will be developed to allow direct observation of the movement of a protein, KORRIGAN1 (KOR1), in plant cells. KOR1 functions in the formation of cellulose fibers, which are the primary constituents of plant cell walls. Cellulose fiber deposition that is controlled by KOR1 influences cell growth and biomass production. KOR1 travels from one compartment to another inside cells before it finally reaches the cell surface where cellulose fibers will ultimately be deposited. To visualize the dynamics of KOR1 in plant cells, this project will apply fluorescent-timer (FT) technology, in which each KOR1 protein is labeled with a time-sensitive fluorescent tag. Because FT changes its color over time, fluorescence images of cells expressing FT-tagged KOR1 will allow visualization of the age and location of KOR1 inside of the cells. This will reveal the route of KOR1 transport through the plant cell. By observing how KOR1 transport changes under different biological circumstances, these studies will reveal how certain factors influence the paths that proteins travel within cells. This is a novel research tool/approach in plant biology that can change the way the scientists view proteins in any plant cell.In plants, distribution of KOR1 among different subcellular domains, such as trans-Golgi network (TGN), plasmamembrane (PM), tonoplast (TP), as well as the cell plate in case of the dividing cells, is scrupulously maintained. Dysfunction of KOR1 is often associated with increased targeting to TP. This project aims to visualize the dynamics of KOR1 transport beyond analysis of endpoint locations. Two transport processes relevant to KOR1 biology will be studied. First, transport route/kinetics and a redirection mechanism of de-novo-synthesized KOR1 will be analyzed by tracking the age of KOR1 after induction of protein expression. Next, KOR1 age information will be used to dissect the multiple targeting routes of KOR1 to the cell plate during cell division. These systems will serve as models for further dissection of various factors that quantitatively affect KOR1 transport. The interdisciplinary team will optimize FT technology for plant cell biology as a whole fluorescence analysis package, including FT with characterized in planta maturation time, compatible fluorescent subcellular markers, and automated image-processing pipelines. The experimental platform established in this project will serve as the basis for further investigation of multiple factors that affect KOR1 transport, including protein N-glycosylation pathways and interactions with cellulose synthase complex.